The goal of this project is to develop sensitive and quantitative imaging and cellular approaches for the evaluation of vaginal and rectal mucosal responses to non-vaccine biomedical prevention (nBP) products aimed at preventing acquisition of HIV. A safe and effective nBP strategy could contribute to reducing the incidence of HIV saving millions of lives. While several promising candidate nBPs have emerged in recent years and progressed to clinical trials, a disappointing outcome has been the halting of several such trials due to safety concerns or failure to prevent HIV. These outcomes highlight the need for improved preclinical safety assessment of candidate nBPs, including the need for the sensitive diagnostic methods that can assess the degree of nBP-induced damage in the mucosal barrier and that can detect the presence of inflammatory cells and mediators that may contribute to increased susceptibility to infection. However, there is a critical gap in the availability of advanced tools for monitoring the in vivo mucosal microenvironment in the context of nBP products, particularly in readily accessible animal models that provide a mucosal environment similar to humans in gross and micro-anatomy and scale. Supporting models and technologies that provide an avenue for non-invasive and repeated in vivo assessment of mucosal barrier response to nBP prevention strategies could play an important role in the nBP pipeline, significantly contributing to the development of more safe and effective products. Here, we hypothesize that changes in microstructure and permeability of mucosal barrier can be detected by subcellular and molecular imaging of vaginal and rectal mucosa in vivo, following topical application of nBPs. Our specific aims are to develop and validate novel cellular and molecular imaging techniques for quantitative monitoring and detection of mucosal irritation with sensitivity that is not currently available, and to validate these techniques as a marker of mucosal irritation and injury that can be correlated to inflammation following topical applications of nBP. The expected outcomes include the development of more sensitive epithelial barrier assessment approaches than are currently available for evaluating the effects of nBPs and advancing the use of the sheep model for in vivo assessment of candidate nBPs, thereby contributing to the pipeline for development of safe and effective nBPs.